ProjectSummary TheprocessofEpithelialtoMesenchymalTransition(EMT)isatransdifferentiationeventinwhichepithelialcells switchtheirphenotypetoamesenchymalone,whichfacilitatesindividualcellmigration,cellinvasion,andtissue assembly.Thisdynamicprocessiscriticalduringembryonicdevelopment,and,whencoupledwiththereverse MET process, facilitates correct spatial and temporal development of organs. While EMT is critical for development,itsmisregulationisimplicatedinmanydiseases,includingcardiacfibrosis,cirrhosis,andcancer. The events that initiate EMT are not well understood, but appear to be linked to changes in the intracellular biochemicalsignalingandgeneexpression,solublefactorsection,autocrinesignaling,mechanicalandparacrine signalingbetweenneighboringcells,andmechanicalsignalingbetweentheepithelialsheetandtheunderlying extracellular matrix (ECM). The Conway lab has demonstrated that a drop in force on adherens junctions is necessary for progression of EMT. The Lemmon lab has demonstrated that progression of EMT requires assemblyoftheECMproteinfibronectin(FN)intonewfibrils,whichcontainbindingsitesforseveralpro-EMT growthfactors.OurrecentstudysuggeststhatFNfibrilsdriveEMTbyclusteringthesegrowthfactorsatthecell surface.ThesefindingshaveledustoahypothesisinwhichEMTisinitiatedbydisruptionofforcesonadherens junctions,whichinturnredistributesforcestotheunderlyingmatrix,initiatesFNfibrilassembly,clusteringsoluble signalsthatpromoteEMTatthecellsurface,anddrivingintracellularbiochemicalsignaling.Inthiswork,we will: 1) develop a computational model that predicts junctional forces, matrix forces and matrix assembly, autocrine and paracrine signaling, and epithelial and mesenchymal cell markers, and uses these to predict spatial localization of EMT in an assembling and confluent cell sheet. Our model will start from a recently developedcell-basedmodelofcell-matrixinteractions,usingthepreviouslydevelopedLemmon-Romermodel of traction force prediction, and extend this to predict force redistribution of cell-matrix forces in neighboring epithelialcellsandtheforcesactingonadherensjunctionsineachcell.Wewillcouplethesemechanicalforces withamodelofintracellularsignalingpathways.2)Wewilluseanovelsuiteoftoolstoestablishanexperimental system that can simultaneously measure junctional forces, matrix forces, FN assembly, and EMT markers. Coloniesofepithelialcellswillbegeneratedusingmicrocontactprinting,whichallowsforrepeatablecolonysize andshape.WewillmeasurespatialregulationofEMTmarkerswithinthecolonieswhilealsoquantifyingboth junctional forces (using a FRET-based biosensor) and cell-matrix forces (using microcontact printing and a substratedeflectiontechnique)todeterminerelationshipbetweenforcecomponents,matrixassemblyandEMT signaling.3)WewillpredicttheeffectsofpotentialEMTinhibitorsandactivatorswiththemodel,thenvalidate the predictive capability of the model using the experimental system. Experimental data will be related to computationalsimulationsforbothprocesses.